The efficient coordination of signal transduction at the plasma membrane is dependent on the segregation, or organization, of sets of signaling proteins in specific microdomains. The overall aim of this study is to dissect the function and composition of the plasma membrane microdomains that are involved in Ras signaling pathways. Ras proteins operate as molecular witches in many signal transduction pathways and are frequently mutated in human tumors. The highly homologous Ras isoforms, H-ras, N-ras, and K-ras, generate different signal out puts most likely because their different C-terminal membrane anchors direct the proteins to different microdomains of the plasma membrane. This project will analyze the protein and lipid content of these micro domains using Ras isoforms as molecular markers. Novel biochemical and electron microscopic techniques will be used to examine the plasma membrane distribution of the different Ras isoforms, to compare their distribution to known activators and effectors, and to reconstitute the dynamics of Ras laterals segregation in vitro. The study will also examine to what extent compartmentalized plasma membrane proteins and lipids contribute to Ras microlocalization and function. Transgenic and knock-out mice will be used to define the role of caveolae in H-ras and K-ras function.This description of the precise molecular environment in which specific Ras isoforms operate may identify novel targets for selective chemotherapy. The specific aims of the project are: 1. A proteomic and lipidomic characterization of Ras surface micro domains 2. An electron microscopic visualization and characterization of surface micro domains 3. An investigation of the dynamic regulation of micro domain localization of Ras and Ras-interacting proteins in response to physiological stimuli.